HOYAJM

I need a figure of a SDS-PAGE gel with a couple different purified proteins. I tend to overload gels because I am just checking quality and size and just load "a few microliters". How much (in ug) protein should be loaded per well? Each sample is pure (only 1 protein) and I am looking to get a normal size band that is more intense than the ladder, but not overwhelming.

Thanks

JM

I know that using the same data for two manuscripts is a BIG no-no, but I have a question about a particular situation.

We have already published data on the characterization of Protein X.

I have now characterized Protein Y. I would like to compare the data in a publication (a part of a publication, 10-20% of total data). Is it OK to make a figure with both Protein X & Y data? The Protein X data would be the previously published data.

I am debating whether or not I should only include Protein Y data and constantly reference the Protein X paper, or include both Protein Y & X data and mention that the X data was taken from the previous publication.

Any take on this would be great,

Thanks

I have done overnight in vitro transcription/translation reactions that produce a 200 Kd protein that can not self-cleave (Lane D) or can self-cleave (Lane E). I have attached the gel image.

In lane D (can not cleave itself) I do see a strong band, albeit at a lower MW then expected, but I think it is right.

In lane E I should observe cleaved proteins (5-6) and a few intermediates, instead I get a smear with only one band visible (about 1/3 down from the top)

I ran a 4-20% HEPES gel from Pierce. I use SDS-HEPES buffer. I have treated all samples with 2X beta mercaptoethanol SDS loading buffer, heated at 70C for 10 minutes, and ran for 2 hrs at 30 milliamps. I exposed for 90 minutes (overnight does not show additional bands)

Can anybody explain the smears? I should be seeing bands from 20-60 Kd in lane E (possibly intermediates at higher MW).

I used the TnT Quick Coupled transcription/translation kit from Promega. The lysate is very viscous and is difficult to run on gels. If anybody has experience with the Promega TnT kit, please let me know.

In short, I really need to improve the quality of these gels with better resolution. My constructs have been sequenced and they should be fine for the TnT reaction. There should be one band in lane D, and around 6 bands in lane E. I am hoping the additional smears in lane E contain the desired proteins and I am simply running the gels incorrectly, somehow.

Thanks for any advice.

JM

I want to look for a specific motif in well-known host-cell proteins that are involved in transcription, translation, even signalling. Ideally I would like to search all of these proteins for a specific ~5 amino acid sequence at once. Where can I get the sequence of such proteins? Is there a specific site that would have most well-known host cell protein sequences?

Please share any websites, tips, etc.

Thanks,

JM

I am attempting to change 18bp in a 10.5kb plasmid. I have designed four primers for the overlap PCR. I have an overlap region of 18bp (the actual sequence I am attempting to change) in these primers.

The primers give two products at expected sizes and the overlap PCR yielded a product at the correct size (piece 1 + piece 2)

My problem is after I digest my vector and overlap PCR product and then ligate is all of my colonies have the wild type sequence. I have used overlap PCR with this same strategy before with no problems. Is it possible to amplify the WT sequence with mutating primers? The only thing I can think of is the digest of my vector was incomplete (1 bad enzyme) and I am just sequencing colonies that self-ligated. I might do a sequential digestion instead of a double digest to test this theory.

Has anyone had any experience with this type of issue. I can dive into specifics of my experiments if needed.

Thanks